Candidate. Dr. Charles Evans is a junior faculty member at the University of Michigan in the department of Internal Medicine. Dr. Evans is trained as an analytical chemist, and has over 7 years of experience working with LC-MS and GC-MS instrumentation for quantitative analysis of metabolites, proteins and other biological molecules. Over the course of his career, Dr. Evans has been extensively involved in instrumentation design and optimization, assay and method development, proteomic and metabolomic studies, and mass isotopomer analysis for metabolic flux analysis. Dr. Evans has maintained a career-long interest in the biological systems which he has frequently studied as an application for the analytical methodology he has developed. Through the research and training proposed in this application, Dr. Evans seeks to become established as an independent investigator in the field of metabolism; in this position he will develop and use novel analytical methods to tackle major issues in the field of metabolic disease. Environment. The University of Michigan is an excellent location to implement the training and research proposed in this application due to the great breadth of basic and clinical research it houses spanning a wide variety of biomedical disciplines. The candidate's mentor, Dr. Charles Burant, is Professor of Metabolism in the Department of Internal Medicine at Michigan. Dr. Burant has extensive experience in basic and translational research in nutrition and metabolic diseases with a career-long interest in the relationship between nutrients and the development of insulin resistance. Dr. Burant is PI on 3 NIH grants and has previously mentored numerous Ph.D. graduate students, postdoctoral fellows, and junior faculty. The co- mentor, Dr. Gregory Cartee, is Professor in the School of Kinesiology. His research is focused on skeletal muscle metabolism especially related to the modulation of glucose transport by exercise and calorie restriction. Dr. Cartee's lab uses in vitro muscle contraction in studies as a model for exercise, and will support Dr. Evans' training in these protocols. Research. In humans, oxidative capacity is a well-established measure of metabolic health. Low oxidative capacity is prevalent in people with the metabolic syndrome, obesity and type 2 diabetes. As the ultimate site of oxygen reduction, mitochondria are key to overall oxidative capacity. Diminished mitochondrial metabolism is seen in populations at high risk for type 2 diabetes and obesity, suggesting a genetic component to oxidative capacity. Reduced mitochondrial mass along with intrinsic changes in carbohydrate and lipid oxidation have been described in obesity and diabetes. However, other evidence suggests that acquired changes in mitochondrial metabolism could lead to many of the defects described in muscle metabolism. Moreover, alterations in mitochondria could be both genetically determined and amplified by environmental factors. What is still unclear is the specific nature of the alterations in metabolism that occur as a result of the genetic predisposition and environmental challenges. To help understand the causes of the metabolic syndrome, a line of low capacity runner (LCR) and high capacity runner (HCR) rats developed by a program of artificial selection for exercise endurance will be investigated. Many of the phenotypic characteristics of the LCR animals parallel those seen in humans with metabolic syndrome, including relative obesity, dyslipidemia as well as reduced oxidative capacity, mitochondrial biogenesis rates, and mitochondrial mass compared to the HCR line. In these studies, in vivo and in vitro metabolomics will be used to measure steady state levels and flux of central carbon metabolism to fully understand the mechanisms that underlie the enhanced oxidative capacity of the HCR compared to the LCR rats. We hypothesize that differences in the balance between anaplerotic and cataplerotic flux of TCA cycle intermediates are fundamental to the difference in oxidative capacity of the LCR and HCR animals and are central to the connection between low oxidative capacity and the metabolic syndrome. To test this hypothesis, quantitative targeted metabolomic assays will be performed in skeletal muscle and plasma of LCR and HCR running rats at rest and during exercise. Next, an in vitro contraction protocol will be established to probe the effects of selected metabolites on exercise endurance of skeletal muscle of LCR and HCR rats. Finally, metabolic flux will be quantified in skeletal muscle from LCR and HCR rats using stable isotope tracers. When complete, these studies will help illuminate the alterations in fuel utilization in a genetically defined model of obesity and the metabolic syndrome. The studies will also provide improved techniques for assessment of metabolic flux in mammalian tissue which will be of benefit to a wide range of future studies in both animals and humans.